The flawless execution of cell division is essential to the generation and survival of all organisms. During every cell cycle, chromosomes must be accurately partitioned to daughter cells to prevent genomic instability and aneuploidy, a hallmark of all tumors and many birth defects. We are studying chromosome segregation in budding yeast because it is amenable to both genetic and biochemical analyses, and the mechanism of chromosome segregation is fundamentally conserved. Chromosomes segregate using their kinetochores, the specialized protein structures that are assembled on centromeric DNA sequences and mediate attachment to the spindle. Because centromeric DNA sequences are not conserved, centromere identity is propagated by an epigenetic mechanism. A hallmark of epigenetic regulation is a specialized chromatin structure that is characterized by the presence of histone variants and unique post-translational modifications to histones. Consistent with this, all eukaryotic centromeres contain an essential histone H3 variant (CenH3) that is required for kinetochore assembly and is likely to be the epigenetic mark that specifies centromere identity. In addition, the H2A.Z histone variant localizes to pericentromeric and centromeric chromatin in a number of organisms, and centromeres contain a distinct histone modification pattern relative to euchromatin. Although a specialized chromatin structure is essential for centromere identity and function, the mechanisms that assemble and maintain centromeric chromatin have not been elucidated. We will therefore take complementary biochemical and genetic approaches to identify factors that deposit CenH3 at centromeres. In addition, we will identify the mechanisms that maintain CenH3 at the centromere and prevent it from localizing to euchromatin. Finally, we have developed a method to enrich for histones associated with centromeres that will allows us to identify centromere-specific histone modifications that are important for chromosome segregation. Taken together, these studies will lead to a better understanding of chromosome segregation and the maintenance of centromere identity and genomic stability in all eukaryotes. Project Narrative All cells must inherit the right number of chromosomes every time they divide because the wrong number of chromosomes is a hallmark of all cancers and a number of birth defects. We are therefore studying the process of chromosome partitioning to daughter cells when they divide to understand the basis for a number of human diseases.